Il-2 muteins and uses thereof

ABSTRACT

The present application provides for IL-2 muteins, compositions comprising the same, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/109,897, filed Aug. 23, 2018, which claims priority to U.S.Provisional Application No. 62/675,972 filed May 24, 2018, and U.S.Provisional Application No. 62/595,357 filed Dec. 6, 2017, each of whichare hereby incorporated by reference in their entirety.

FIELD

Embodiments provided herein relate to proteins referred to as IL-2muteins, compositions comprising the same, and methods of using thesame.

BACKGROUND

IL-2 binds three transmembrane receptor subunits: IL-2Rβ and IL-2Rγ,which together activate intracellular signaling events upon IL-2binding, and CD25 (IL-2Rα) which serves to present IL-2 to the other 2receptor subunits. The signals delivered by IL-2Rβγ include those of thePI3-kinase, Ras-MAP-kinase, and STAT5 pathways.

T cells require expression of CD25 to respond to the low concentrationsof IL-2 that typically exist in tissues. T cells that express CD25include both CD4⁺ FOXP3⁺ regulatory T cells (T-reg cells)—which areessential for suppressing autoimmune inflammation—and FOXP3⁻ T cellsthat have been activated to express CD25. FOXP3⁻ CD4⁺ T effector cells(T-eff) may be either CD4⁺or CD8⁺ cells, both of which can bepro-inflammatory and may contribute to autoimmunity and other diseaseswhere the subject's immune system attacks an organ or other tissues.IL-2-stimulated STAT5 signaling is crucial for normal T-reg cell growthand survival and for high FOXP3 expression.

Because of the low affinity IL-2 possesses for each of the three IL-2Rchains, a further reduction in affinity for IL-2Rβ and IL-2Rγ could beoffset by an increased affinity for CD25. Mutational variants of IL-2have been generated. These IL-2 mutants can be referred to as IL-2muteins and have been found useful in the treatment of various diseases.However, there is still a need for additional IL-2 muteins that can beused in various applications and compositions. The present embodimentssatisfies these needs as well as others.

SUMMARY

In some embodiments, peptides comprising an amino acid sequence of SEQID NO: 1, wherein the peptide comprises a mutation at position 73, 76,100, or 138 are provided.

In some embodiments peptides comprising an amino acid sequence of SEQ IDNO: 2, wherein the peptide comprises a mutation at position 53, 56, 80,or 118 are provided.

Also provided are pharmaceutical compositions comprising the same andnucleic acid molecules encoding the proteins described herein. Alsoprovided herein are vectors comprising the nucleic acid moleculeencoding the proteins described herein. In some embodiments, plasmidscomprising the nucleic acid encoding the proteins described herein areprovided. In some embodiments, cells comprising the nucleic acidmolecules, vectors, or plasmids, encoding the proteins described hereinare provided.

In some embodiments, methods of activating T regulatory cells areprovided. In some embodiments, the methods comprise contacting a Tregulatory cell with a peptide described herein or a pharmaceuticalcomposition described herein.

In some embodiments, methods of treating an inflammatory disorder in asubject are provided. In some embodiments, the methods compriseadministering to a subject, including but not limited to a subject inneed thereof, a peptide (e.g. a therapeutically effective amount of thepeptide).

In some embodiments, methods of promoting or stimulating STAT5phosphorylation in T regulatory cells are provided. In some embodiments,the methods comprise administering to a subject a peptide (e.g. atherapeutically effective amount of the peptide).

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates a non-limiting embodiment of a IL-2 mutein providedfor herein.

DETAILED DESCRIPTION

Described herein are therapeutics that can modulate (e.g. increase)T-reg cell proliferation, survival, activation and/or function. In someembodiments, the modulation is selective or specific for the T-regcells.

As used herein, the term “selective” refers to the therapeutic orprotein modulating the activity in T-reg cells but has limited or lacksthe ability to promote the activity in non-regulatory T cells.

In some embodiments, the therapeutic is a mutant of IL-2. A mutant ofIL-2 can be referred to as an IL-2 mutein. IL-2 can exist in twodifferent forms, an immature form and a mature form. The mature form iswhere the leader sequence has been removed. This is done during apost-translational process. The wild-type sequence of the immature IL-2is as follows:

-   -   MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNY        KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNFHLR        PRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:        1).        The wild-type sequence of the mature IL-2 is as follows:    -   APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA        TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETT        FMCEYADETATIVEFLNRWITFCQSIISTLT (mature IL-2 sequence) (SEQ ID        NO: 2).

An IL-2 mutein molecule can be prepared by mutating one or more of theresidues of IL-2. Non-limiting examples of IL-2-muteins can be found inWO2016/164937, U.S. Pat. No. 9,580,486, U.S. Pat. No. 7,105,653, U.S.Pat. No. 9,616,105, U.S. Pat. No. 9,428,567, US2017/0051029,US2014/0286898A1, WO2014153111A2, WO2010/085495, WO2016014428A2,WO2016025385A1, and US20060269515, each of which are incorporated byreference in its entirety.

In some embodiments, the alanine at position 1 of the sequence above(SEQ ID NO: 2) is deleted. In some embodiments, the IL-2 mutein moleculecomprises a serine substituted for cysteine at position 125 of themature IL-2 sequence. Other combinations of mutations and substitutionsthat are IL-2 mutein molecules are described in US20060269515, which isincorporated by reference in its entirety. In some embodiments, thecysteine at position 125 is also substituted with a valine or alanine.In some embodiments, the IL-2 mutein molecule comprises a V91Ksubstitution. In some embodiments, the IL-2 mutein molecule comprises aN88D substitution. In some embodiments, the IL-2 mutein moleculecomprises a N88R substitution. In some embodiments, the IL-2 muteinmolecule comprises a substitution of H16E, D84K, V91N, N88D, V91K, orV91R, any combinations thereof. In some embodiments, these IL-2 muteinmolecules also comprise a substitution at position 125 as describedherein. In some embodiments, the IL-2 mutein molecule comprises one ormore substitutions selected from the group consisting of: T3N, T3A,L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K,H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N,L19R, L19S, L19T, L19V, D20A, D20E, D20H, D20I, D20Y, D20F, D20G, D20T,D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84QD84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R,N88S, N88V, N88W, V91D, V91E, V91G, V91S, 192K, I92R, E95G, and Q126. Insome embodiments, the amino acid sequence of the IL-2 mutein moleculediffers from the amino acid sequence set forth in mature IL-2 sequencewith a C125A or C125S substitution and with one substitution selectedfrom T3N, T3A, L12G, L12K, L12Q L12S, Q13G, E15A, E15G, E15S, H16A,H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D,L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T,D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q,D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R,N88S, N88V, N88W, V91D, V91E, V91G, V91S, 192K, I92R, E95G, Q126I,Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule differsfrom the amino acid sequence set forth in mature IL-2 sequence with aC125A or C125S substitution and with one substitution selected fromD20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R,H16T, H16V, 192K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D,V91G, V91K, and V91S. In some embodiments, the IL-2 mutein comprisesN88R and/or D20H mutations.

In some embodiments, the IL-2 mutein molecule comprises a mutation inthe polypeptide sequence at a position selected from the groupconsisting of amino acid 30, amino acid 31, amino acid 35, amino acid69, and amino acid 74. In some embodiments, the mutation at position 30is N30S. In some embodiments, the mutation at position 31 is Y31H. Insome embodiments, the mutation at position 35 is K35R. In someembodiments, the mutation at position 69 is V69A. In some embodiments,the mutation at position 74 is Q74P. In some embodiments, the muteindoes not comprise a mutation at position 30, 31, and/or 35.

In some embodiments, the IL-2 mutein molecule comprises a substitutionselected from the group consisting of: N88R, N88I, N88G, D20H, D109C,Q126L, Q126F, D84G, or D84I relative to mature human IL-2 sequenceprovided above. In some embodiments, the IL-2 mutein molecule comprisesa substitution of D109C and one or both of a N88R substitution and aC125S substitution. In some embodiments, the cysteine that is in theIL-2 mutein molecule at position 109 is linked to a polyethylene glycolmoiety, wherein the polyethylene glycol moiety has a molecular weight offrom about 5 to about 40 kDa. In some embodiments, the mutein does notcomprise a mutation at position 109, 126, or 84.

In some embodiments, any of the substitutions described herein arecombined with a substitution at position 125. The substitution can be aC125S, C125A, or C125V substitution. In some embodiments, the muteindoes not comprise a mutation at position 125.

The numbering referred to herein, unless indicated otherwise for theIL-2 muteins refers to the mature sequence. If a sequence or positionrefers to SEQ ID NO: 1 it is the immature sequence. However, totranspose the positions from the immature sequence (SEQ ID NO: 1) to themature sequence (SEQ ID NO: 2) all that need be done is to subtract 20from the position referred to in SEQ ID NO: 1 to get the correspondingposition in SEQ ID NO: 2.

In addition to the substitutions or mutations described herein, in someembodiments, the IL-2 mutein has a substitution/mutation at one or moreof positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 orpositions at one or more of positions 53, 56, 80, or 118 that correspondto SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises amutation at positions 73 and 76; 73 and 100; 73 and 138; 76 and 100; 76and 138; 100 and 138; 73, 76, and 100; 73, 76, and 138; 73, 100, and138; 76, 100 and 138; or at each of 73, 76, 100, and 138 that correspondto SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises amutation at positions 53 and 56; 53 and 80; 53 and 118; 56 and 80; 56and 118; 80 and 118; 53, 56, and 80; 53, 56, and 118; 53, 80, and 118;56, 80 and 118; or at each of 53, 56, 80, and 118 that correspond to SEQID NO: 2. As the IL-2 can be fused or tethered to other proteins, asused herein, the term corresponds to as reference to a SEQ ID NOs: 6 or15 refer to how the sequences would align with default settings foralignment software, such as can be used with the NCBI website. In someembodiments, the mutation is leucine to isoleucine. Thus, the IL-2mutein can comprise one more isoleucines at positions 73, 76, 100, or138 that correspond to SEQ ID NO: 1 or positions at one or more ofpositions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2. In someembodiments, the mutein comprises a mutation at L53 that correspond toSEQ ID NO: 2. In some embodiments, the mutein comprises a mutation atL56 that correspond to SEQ ID NO: 2. In some embodiments, the muteincomprises a mutation at L80 that correspond to SEQ ID NO: 2. In someembodiments, the mutein comprises a mutation at L118 that correspond toSEQ ID NO: 2. In some embodiments, the mutation is leucine toisoleucine. In some embodiments, the mutein also comprises a mutation asposition 69, 74, 88, 125, or any combination thereof in these muteinsthat correspond to SEQ ID NO: 2. In some embodiments, the mutation is aV69A mutation. In some embodiments, the mutation is a Q74P mutation. Insome embodiments, the mutation is a N88D or N88R mutation. In someembodiments, the mutation is a C125A or C125S mutation.

In some embodiments, the IL-2 mutein comprises a mutation at one more ofpositions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145 that correspondto SEQ ID NO: 1 or one or more positions 29, 31, 35, 37, 48, 69, 71, 74,88, and 125 that correspond to SEQ ID NO: 2. The substitutions can beused alone or in combination with one another. In some embodiments, theIL-2 mutein comprises substitutions at 2, 3, 4, 5, 6, 7, 8, 9, or eachof positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145. Non-limitingexamples such combinations include, but are not limited to, a mutationat positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145; 49, 51, 55,57, 68, 89, 91, 94, and 108; 49, 51, 55, 57, 68, 89, 91, and 94; 49, 51,55, 57, 68, 89, and 91; 49, 51, 55, 57, 68, and 89; 49, 51, 55, 57, and68; 49, 51, 55, and 57; 49, 51, and 55; 49 and 51; 51, 55, 57, 68, 89,91, 94, 108, and 145; 51, 55, 57, 68, 89, 91, 94, and 108; 51, 55, 57,68, 89, 91, and 94; 51, 55, 57, 68, 89, and 91; 51, 55, 57, 68, and 89;55, 57, and 68; 55 and 57; 55, 57, 68, 89, 91, 94, 108, and 145; 55, 57,68, 89, 91, 94, and 108; 55, 57, 68, 89, 91, and 94; 55, 57, 68, 89, 91,and 94; 55, 57, 68, 89, and 91; 55, 57, 68, and 89; 55, 57, and 68; 55and 57; 57, 68, 89, 91, 94, 108, and 145; 57, 68, 89, 91, 94, and 108;57, 68, 89, 91, and 94; 57, 68, 89, and 91; 57, 68, and 89; 57 and 68;68, 89, 91, 94, 108, and 145; 68, 89, 91, 94, and 108; 68, 89, 91, and94; 68, 89, and 91; 68 and 89; 89, 91, 94, 108, and 145; 89, 91, 94, and108; 89, 91, and 94; 89 and 91; 91, 94, 108, and 145; 91, 94, and 108;91, and 94; or 94 and 108. Each mutation can be combined with oneanother. The same substitutions can be made in SEQ ID NO: 2, but thenumbering would adjusted appropriately as is clear from the presentdisclosure (20 less than the numbering for SEQ ID NO: 1 corresponds tothe positions in SEQ ID NO: 2).

In some embodiments, the IL-2 mutein comprises a mutation at one or morepositions of 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO:1 or the equivalent positions at SEQ ID NO: 2 (e.g. positions 15, 16,22, 84, 95, and 126). These mutations can be combined with the otherleucine to isoleucine mutations described herein or the mutation atpositions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 or at oneor more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2.In some embodiments, the mutation is a E35Q, H36N, Q42E, D104N, E115Q,or Q146E, or any combination thereof. In some embodiments, one or moreof these substitutions is wildtype. In some embodiments, the muteincomprises a wild-type residue at one or more of positions 35, 36, 42,104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalentpositions at SEQ ID NO: 2 (e.g. positions 15, 16, 22, 84, 95, or 126).

The mutations at these positions can be combined with any of the othermutations described herein, including, but not limited to substitutionsat positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 orpositions at one or more of positions 53, 56, 80, or 118 that correspondto SEQ ID NO: 2 described herein and above. In some embodiments, theIL-2 mutein comprises a N49S mutation that corresponds to SEQ ID NO: 1.In some embodiments, the IL-2 mutein comprises a Y51S or a Y51H mutationthat corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 muteincomprises a K55R mutation that corresponds to SEQ ID NO: 1. In someembodiments, the IL-2 mutein comprises a T57A mutation that correspondsto SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a K68Emutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2mutein comprises a V89A mutation that corresponds to SEQ ID NO: 1. Insome embodiments, the IL-2 mutein comprises a N91R mutation thatcorresponds to SEQ ID NO: 1. In some embodiments, the IL-2 muteincomprises a Q94P mutation that corresponds to SEQ ID NO: 1. In someembodiments, the IL-2 mutein comprises a N108D or a N108R mutation thatcorresponds to SEQ ID NO: 1. In some embodiments, the IL-2 muteincomprises a C145A or C145S mutation that corresponds to SEQ ID NO: 1.

These substitutions can be used alone or in combination with oneanother. In some embodiments, the mutein comprises each of thesesubstitutions. In some embodiments, the mutein comprises 1, 2, 3, 4, 5,6, 7, or 8 of these mutations. In some embodiments, the mutein comprisesa wild-type residue at one or more of positions 35, 36, 42, 104, 115, or146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQID NO: 2 (e.g. positions 15, 16, 22, 84, 95, 126, and 126).

In some embodiments, the IL-2 mutein comprises a N29S mutation thatcorresponds to SEQ ID NO: 2. In some embodiments, the IL-2 muteincomprises a Y31S or a Y31H mutation that corresponds to SEQ ID NO: 2. Insome embodiments, the IL-2 mutein comprises a K35R mutation thatcorresponds to SEQ ID NO: 2. In some embodiments, the IL-2 muteincomprises a T37A mutation that corresponds to SEQ ID NO: 2. In someembodiments, the IL-2 mutein comprises a K48E mutation that correspondsto SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a V69Amutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2mutein comprises a N71R mutation that corresponds to SEQ ID NO: 2. Insome embodiments, the IL-2 mutein comprises a Q74P mutation thatcorresponds to SEQ ID NO: 2. In some embodiments, the IL-2 muteincomprises a N88D or a N88R mutation that corresponds to SEQ ID NO: 2. Insome embodiments, the IL-2 mutein comprises a C125A or C125S mutationthat corresponds to SEQ ID NO: 2. These substitutions can be used aloneor in combination with one another. In some embodiments, the muteincomprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In someembodiments, the mutein comprises each of these substitutions. In someembodiments, the mutein comprises a wild-type residue at one or more ofpositions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1or the equivalent positions at SEQ ID NO: 2 (e.g. positions 15, 16, 22,84, 95, and 126).

For any of the IL-2 muteins described herein, in some embodiments, oneor more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQID NO: 1 or the equivalent positions at SEQ ID NO: 2 (e.g. positions 15,16, 22, 84, 95, and 126) are wild-type (e.g. are as shown in SEQ ID NOs:1 or 2). In some embodiments, 2, 3, 4, 5, 6, or each of positions 35,36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or theequivalent positions at SEQ ID NO: 2 (e.g. positions 15, 16, 22, 84, 95,and 126) are wild-type.

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 3) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS TLT

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 4) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS TLT

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 5) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS TLT

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 6) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIIS TLT

In some embodiments, the IL-2 mutein sequences described herein do notcomprise the IL-2 leader sequence. The IL-2 leader sequence can berepresented by the sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).Therefore, in some embodiments, the sequences illustrated above can alsoencompass peptides without the leader sequence. Although SEQ ID NOs; 3-6are illustrated with only mutation at one of positions 73, 76, 100, or138 that correspond to SEQ ID NO: 1 or positions at one or more ofpositions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2, thepeptides can comprise 1, 2, 3, or 4 of the mutations at these positions.In some embodiments, the substitution at each position is isoleucine orother type of conservative amino acid substitution. In some embodiments,the leucine at the recited positions are substituted with,independently, isoleucine, valine, methionine, or glycine, alanine,glutamine or glutamic acid.

In some embodiments, the IL-2 protein of SEQ ID NO: 2 comprises thefollowing mutations: V69A, Q74P, N88D, and C125S or C125A and onemutation selected from the group consisting of L53I, L56I, L80I, andL118I. In some embodiments, the IL-2 protein comprises two mutationsselected from the group consisting of L53I, L56I, L80I, and L118I. Insome embodiments, the IL-2 protein comprises three or each of themutations selected from the group consisting of L53I, L56I, L80I, andL118I. In some embodiments, the IL-2 protein comprises L53I and L56I,L53I and L80I, L53I and L118I, L56I and L80I, L56I and L118I, L80I andL118I, L53I, L56I, and L80I, L53I, L56I, and L118I, L56I, L80I, andL118I or L53I, L56I, L80I, and L118I. In some embodiments, the IL-2mutein does not comprise L53I, L56I, L80I, or L118I mutations. In someembodiments, the IL-2 mutein comprises a T3A mutation.

In some embodiments, the IL-2 protein of SEQ ID NO: 2 comprises thefollowing mutations: V69A, Q74P, N88D, and C125S or C125A and one ormore mutations, such as but not limited to conservative substitutions,in regions of 45-55, 50-60, 52-57, 75-85, 100-130, 115-125 of SEQ ID NO:2.

In some embodiments, the IL-2 mutein molecule is fused to a Fc Region orother linker region as described herein. Examples of such fusionproteins can be found in U.S. Pat. No. 9,580,486, U.S. Pat. No.7,105,653, U.S. Pat. No. 9,616,105, U.S. Pat. No. 9,428,567,US2017/0051029, WO2016/164937, US2014/0286898A1, WO2014153111A2,WO2010/085495, WO2016014428A2, WO2016025385A1, US2017/0037102, andUS2006/0269515, each of which are incorporated by reference in itsentirety.

In some embodiments, the Fc Region comprises what is known at the LALAmutations. In some embodiments, the Fc region comprises L234A and L235Amutations (EU numbering). In some embodiments, the Fc Region comprises aG237A (EU numbering). In some embodiments, the Fc Region does notcomprise a mutation at position G237 (EU numbering) Using the Kabatnumbering this would correspond to L247A, L248A, and/or G250A. In someembodiments, using the EU numbering system the Fc region comprises aL234A mutation, a L235A mutation, and/or a G237A mutation. Regardless ofthe numbering system used, in some embodiments, the Fc portion cancomprise mutations that corresponds to one or more of these residues. Insome embodiments, the Fc Region comprises N297G or N297A (kabatnumbering) mutations. The Kabat numbering is based upon a full-lengthsequence, but would be used in a fragment based upon a traditionalalignment used by one of skill in the art for the Fc region (see, forexample, Kabat et al. (“Sequence of proteins of immunological interest,”US Public Health Services, NIH Bethesda, Md., Publication No. 91, whichis hereby incorporated by reference), which is hereby incorporated byreference. In some embodiments, the Fc Region comprises a sequence of:

(SEQ ID NO: 8) DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.In some embodiments, the Fc Region comprises a sequence of:

(SEQ ID NO: 15) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, the IL-2 mutein is linked to the Fc Region.Non-limiting examples of linkers are glycine/serine linkers. Forexample, a glycine/serine linker can be, or comprise, a sequence ofGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9) or be, or comprise a sequence ofGGGGSGGGGSGGGGS (SEQ ID NO: 16). This is simply a non-limiting exampleand the linker can have varying number of GGGGS (SEQ ID NO: 10) repeats.In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 of the GGGGS (SEQ ID NO: 10) repeats.

In some embodiments, the IL-2 mutein is linked to the Fc Region using aflexible, rigid or cleavable linker. The linker can be as describedherein or as illustrated in the following table:

Type Sequence flexible GGGGS flexible (GGGGS)₃ flexible (GGGGS)_(n) (n =1, 2, 3, 4) flexible (Gly)₈ flexible (Gly)₆ rigid (EAAAK)₃ rigid(EAAK)_(n) (n = 1-3) rigid A(EAAAK)₄ALEA(EAAAK)₄A rigid AEAAAKEAAAKArigid PAPAP rigid (Ala-Pro)_(n) (10-34 aa) cleavable disulfide cleavableVSQTSKLTRAETVFPDV cleavable PLGLWA cleavable RVLAEA cleavable EDVVCCSMSYcleavable GGIEGRGS cleavable TRHRQPRGWE cleavable AGNRVRRSVG cleavableRRRRRRRRR cleavable GFLG Dipeptide LE

Thus, the IL-2/Fc Fusion can be represented by the formula ofZ_(IL-2M)-L_(gs)-Z_(Fc), wherein Z_(IL-2M) is an IL-2 mutein asdescribed herein, L_(gs) is a linker sequence as described herein (e.g.glycine/serine linker) and Z_(Fc) is a Fc region described herein orknown to one of skill in the art. In some embodiments, the formula canbe in the reverse orientation Z_(Fc)-L_(gs)-Z_(IL-2M).

In some embodiments, the IL-2/Fc fusion comprises a sequence of:

(SEQ ID NO: 11) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, the IL-2/Fc fusion comprises a sequence of:

(SEQ ID NO: 12) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, the IL-2/Fc fusion comprises a sequence of:

(SEQ ID NO: 13) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, the IL-2/Fc fusion comprises a sequence of:

(SEQ ID NO: 14) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

In some embodiments, the Fc region of SEQ ID NO: 8 is replaced with SEQID NO: 15.

The proteins described herein can also be fused to another protein, suchas an antibody or other type of therapeutic molecule.

In some embodiments, the sequence of IL-2 mutein or IL-2/Fc fusion areas shown in the following table:

SEQ ID Brief NO: Description Amino Acid Sequence 17 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with C125SCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI mutationVEFLNRWITFSQSIISTLT 18 Human IL-2APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with C125SCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI and T3AVEFLNRWITFSQSIISTLT mutations 19 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with N88R CLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATI and C125SVEFLNRWITFSQSIISTLT 20 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI Q74P andVEFLNRWITFSQSIISTLT C125S mutations 21 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P, N88DVEFLNRWITFSQSIISTLT and C125S mutations 22 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATI Q74P, N88RVEFLNRWITFSQSIISTLT and C125S mutations 23 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with N88D CLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI and C125SVEFLNRWITFSQSIISTLT 24 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEIKHLQ with L53I,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI V69A, Q74P,VEFLNRWITFSQSIISTLT N88D and C125S mutations 25 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHIQ with L56I,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI V69A, Q74P,VEFLNRWITFSQSIISTLT N88D and C125S mutations 26 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with V69A,CLEEELKPLEEALNLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P, L80I,VEFLNRWITFSQSIISTLT N88D and C125S mutations 27 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ with V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P, N88D,VEFINRWITFSQSIISTLT L118I, and C125S mutations 28 Human DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW IgG1 FcYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK (N-terminalTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY fusions) KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with L234A,L235A, and G237A mutations 29 Human DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW IgG1 FcYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK (truncated)TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY with N297GKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG mutation 30 IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ C125S-CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI G4Sx3-FcVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 31 IL-2 T3A,APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ C125S-CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI G4Sx3-FcVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 32 IL-2 N88R,APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ C125S-CLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATI G4Sx3-FcVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 33 IL-2 V69A,APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ Q74P,CLEEELKPLEEALNLAPSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI C125S,-VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLF G4Sx3-FcPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 34 IL-2 N88DAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ V69A, Q74P,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI C125S-VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLF G4Sx3-FcPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 35 IL-2 N88RAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ V69A, Q74P,CLEEELKPLEEALNLAPSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATI C125S-VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLF G4Sx3-FcPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 36 IL-2 N88D,APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ C125S-CLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI G4Sx3-FcVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 37 IL-2 L53I,APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEIKHLQ N88D, V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P, C125S-VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAP G4Sx4-FcSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 38 IL-2 L56IAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHIQ N88D, V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P, C125S-VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAP G4Sx4-FcSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 39 IL-2 L80IAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ N88D V69A,CLEEELKPLEEALNLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P,VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAP C125S-SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ G4Sx4-FcYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 40 IL-2 L118IAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ N88D V69A,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI Q74P,VEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAP C125S-SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ G4Sx4-FcYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 41 IL-2 N88DAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ V69A, Q74P,CLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI C125S-VEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAP G4Sx4-FcSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 42 Fc-G4S-DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW IL-2YVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK N88D V69A,TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY Q74PKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

Each of the proteins may also be considered to have the C125S and theLALA and/or G237A mutations as provided for herein. The C125substitution can also be C125A as described throughout the presentapplication.

In some embodiments, the sequences shown in the table or throughout thepresent application comprise or don't comprise one or more mutationsthat correspond to positions L53, L56, L80, and L118. In someembodiments, the sequences shown in the table or throughout the presentapplication comprise or don't comprise one or more mutations thatcorrespond to positions L59I, L63I, I24L, L94I, L96I or L132I or othersubstitutions at the same positions. In some embodiments, the mutationis leucine to isoleucine. In some embodiments, the mutein does notcomprise another mutation other than as shown or described herein. Insome embodiments, the peptide comprises a sequence of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,or SEQ ID NO: 42.

In some embodiments, the Fc portion of the fusion is not included. Insome embodiments, the peptide consists essentially of an IL-2 muteinprovided for herein. In some embodiments, the protein is free of a Fcportion.

In some embodiments, the IL-2 mutein can be in the format as illustratedin FIG. 1. But as described herein, the IL-2 mutein can, in someembodiments, be used without a Fc domain or the Fc-domain is linked tothe N-terminus of the IL-2 mutein as opposed to the Fc domain beinglinked to the C-terminus of the IL-2 mutein. The polypeptides describedherein also encompass variants of the peptides described. In someembodiments, the IL-2 variants comprise a sequence of amino acids atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93% at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% substantiallysimilar to the sequences provided for herein. The variants include thosethat are described herein with the various substitutions describedherein and above. In some embodiments, the variant has 1, 2, 3, 4, or 5additional substitutions. In some embodiments, the substitution is G toA, L to I, G to S K to R, or other types of conservative substitutions.In some embodiments, the conservative substitution is selected basedupon the following tables:

Basic (positively arginine charged R-groups): lysine histidine Acidic(negatively glutamic acid charged R-groups): aspartic acid Polar(Uncharged R- glutamine groups): asparagine serine threonine cysteineproline Non-Polar (aliphatic R- glycine groups): alanine valinemethionine leucine isoleucine Non-Polar (aromatic R- phenylalaninegroups): tryptophan tyrosine

Original Residue Substitutions Ala Gly; Ser; Thr Arg Lys; Gln Asn Gln;His; Ser Asp Glu; Asn Cys Ser, Sec Gln Asn; Ser; Asp; Glu Glu Asp; Gln;Lys Gly Ala; Pro; Asn His Asn; Gln; Tyr; Phe Ile Leu; Val; Met; Phe LeuIle; Val; Met; Phe Lys Arg; Gln; Met Leu; Tyr; Ile; norleucine; Val; PhePro Beta homo proline; Ser; Thr; Ala; Gly; alpha homoproline Phe Met;Leu; Tyr; Trp Ser Thr; Gly; Asn; Asp Thr Ser; Asn Trp Tyr; Phe,; TyrTrp; Phe; Val Ile; Leu; Met; Phe

The percent identity of two amino acid or two nucleic acid sequences canbe determined by visual inspection and mathematical calculation, or forexample, the comparison is done by comparing sequence information usinga computer program. An exemplary computer program is the GeneticsComputer Group (GCG; Madison, Wis.) Wisconsin package version 10.0program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12: 387-95).The preferred default parameters for the GAP program includes: (1) TheGCG implementation of a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted amino acid comparison matrix of Gribskov and Burgess, ((1986)Nucleic Acids Res. 14: 6745) as described in Atlas of PolypeptideSequence and Structure, Schwartz and Dayhoff, eds., National BiomedicalResearch Foundation, pp. 353-358 (1979) or other comparable comparisonmatrices; (2) a penalty of 8 for each gap and an additional penalty of 2for each symbol in each gap for amino acid sequences, or a penalty of 50for each gap and an additional penalty of 3 for each symbol in each gapfor nucleotide sequences; (3) no penalty for end gaps; and (4) nomaximum penalty for long gaps. Other programs used by those skilled inthe art of sequence comparison can also be used.

In some embodiments, the IL-2 muteins provided herein include proteinsthat have altered signaling through certain pathways activated bywild-type IL-2 via the IL-2R and result in preferentialproliferation/survival/activation of T-regs.

The IL-2 muteins provided for herein can be produced using any suitablemethod known in the art, including those described in U.S. Pat. No.6,955,807 for producing IL-2 variants, which is hereby incorporated byreference. Such methods include constructing a DNA sequence encoding theIL-2 variant and expressing those sequences in a suitably transformedhost, such as a host cell. Utilizing these methods will producerecombinant proteins as provided herein. Proteins can also be producedsynthetically or a combination of synthetic and recombinantly producingfragments in a cell and then combining the fragments to make the entireprotein of interest.

In some embodiments, a nucleic acid molecule (e.g. DNA or RNA) isprepared by isolating or synthesizing a nucleic acid molecule encodingthe protein of interest. Alternatively, the wild-type sequence of IL-2can be isolated and the mutated using routine techniques, such assite-specific mutagenesis.

Another method of constructing a DNA sequence encoding the IL-2 variantwould be chemical synthesis. This for example includes direct synthesisof a peptide by chemical means of the protein sequence encoding for anIL-2 variant exhibiting the properties described herein. This method mayincorporate both natural and unnatural amino acids at various positions.Alternatively, a nucleic acid molecule which encodes a desired proteinmay be synthesized by chemical means using an oligonucleotidesynthesizer. The oligonucleotides are designed based on the amino acidsequence of the desired protein, which can also be selected by usingcodons that are favored in the cell in which the recombinant variantwill be produced. It is well recognized that the genetic code isdegenerate—that an amino acid may be coded for by more than one codon.Accordingly, it will be appreciated that for a given DNA sequenceencoding a particular IL-2 protein, there will be many DNA degeneratesequences that will code for that IL-2 variant. According, in someembodiments, a nucleic acid molecule is provided that encodes theproteins described herein. The nucleic acid molecule can be DNA or RNA.

In some embodiments, the nucleic acid molecule will encode a signalsequence. A signal sequence can be chosen based upon the cell that willbe expressed in. In some embodiments, if the host cell is prokaryotic,the nucleic acid molecule does not comprise a signal sequence. In someembodiments, if the host cell is a eukaryotic cell, the signal sequencecan be used. In some embodiments, the signal sequence is the IL-2 signalsequence.

A nucleic acid molecule “encodes” a protein, as meant herein, if thenucleic acid molecule or its complement comprises the codons encodingthe protein.

“Recombinant” as it applies to polypeptides or proteins, means that theproduction of the protein is dependent on at least one step in whichnucleic acids, which may or may not encode the protein, are introducedinto a cell in which they are not naturally found.

Various host (animals or cell systems) can be used to produce theproteins described herein. Examples of suitable host cells include, butare not limited to, bacteria, fungi (including yeasts), plant, insect,mammal, or other appropriate animal cells or cell lines, as well astransgenic animals or plants. In some embodiments, these hosts mayinclude well known eukaryotic and prokaryotic hosts, such as strains ofE. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cellssuch as Spodoptera frugiperda (Sf9), animal cells such as Chinesehamster ovary (CHO) and mouse cells such as NS/O, African green monkeycells such as COS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells,as well as plant cells in tissue culture. For animal cell expression,CHO cells and COS 7 cells in cultures and particularly the CHO cell lineCHO (DHFR-) or the HKB line may be used.

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences described herein. Neither will all hosts function equally wellwith the same expression system. However, one of skill in the art maymake a selection among these vectors, expression control sequences andhosts without undue experimentation. For example, in selecting a vector,the host must be considered because the vector must replicate in it. Thevectors copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. For example, preferredvectors for use in this invention include those that allow the DNAencoding the IL-2 variants to be amplified in copy number. Suchamplifiable vectors are well known in the art.

Vectors and Host Cells

Accordingly, in some embodiments, vectors encoding the proteinsdescribed herein are provided, as well as host cells transformed withsuch vectors. Any nucleic acids encoding the proteins described hereinmay be contained in a vector, which can, for example, comprise aselectable marker and an origin of replication, for propagation in ahost. In some embodiments, the vectors further include suitabletranscriptional or translational regulatory sequences, such as thosederived from a mammalian, microbial, viral, or insect genes, operablylinked to the nucleic acid molecule encoding the protein. Examples ofsuch regulatory sequences include transcriptional promoters, operators,or enhancers, mRNA ribosomal binding sites, and appropriate sequencesthat control transcription and translation. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA encoding the target protein. Thus, a promoter nucleotide sequence isoperably linked to a nucleic acid molecule if the promoter nucleotidesequence directs the transcription of the nucleic acid molecule.

The host cells that can be used here described herein.

Pharmaceutical Compositions

In another aspect, the present embodiments provide compositions, e.g.,pharmaceutically acceptable compositions, which include a therapeuticcompound (IL-2 mutein) described herein, formulated together with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier can be suitable for intravenous,intramuscular, subcutaneous, parenteral, rectal, local, topical, spinalor epidermal administration (e.g. by injection or infusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Typical compositions are in the form of injectable or infusiblesolutions. In an embodiment the mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In anembodiment, the therapeutic molecule is administered by intravenousinfusion or injection. In another embodiment, the therapeutic moleculeis administered by intramuscular or subcutaneous injection. In anotherembodiment, the therapeutic molecule is administered locally, e.g., byinjection, or topical application, to a target site.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high therapeutic molecule concentration.Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., therapeutic molecule) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

As will be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, a therapeutic compound can be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.Therapeutic compositions can also be administered with medical devicesknown in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a therapeutic compound is 0.1-30mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens ofthe therapeutic compound can be determined by a skilled artisan. Incertain embodiments, the therapeutic compound is administered byinjection (e.g., subcutaneously or intravenously) at a dose of about 1to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20mg/kg, 15 to 25 mg/kg, or about 3 mg/kg. The dosing schedule can varyfrom e.g., once a week to once every 2, 3, or 4 weeks, or, in someembodiments, the dosing schedule can be, once every month, every 2months, every 3 months, or every 6 months. In one embodiment, thetherapeutic compound is administered at a dose from about 10 to 20 mg/kgevery other week. The therapeutic compound can be administered byintravenous infusion at a rate of more than 20 mg/min, e.g., 20-40mg/min, and typically greater than or equal to 40 mg/min to reach a doseof about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and moretypically, about 110 to 130 mg/m2. In embodiments, the infusion rate ofabout 110 to 130 mg/m2 achieves a level of about 3 mg/kg. In otherembodiments, the therapeutic compound can be administered by intravenousinfusion at a rate of less than 10 mg/min, e.g., less than or equal to 5mg/min to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50mg/m2, about 7 to 25 mg/m2, or, about 10 mg/m2. In some embodiments, thetherapeutic compound is infused over a period of about 30 min. It is tobe noted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a therapeutic molecule of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of a therapeutic molecule may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the therapeutic compound to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of a therapeuticmolecule t is outweighed by the therapeutically beneficial effects. A“therapeutically effective dosage” preferably inhibits a measurableparameter, e.g., immune attack at least about 20%, more preferably by atleast about 40%, even more preferably by at least about 60%, and stillmore preferably by at least about 80% relative to untreated subjects.The ability of a compound to inhibit a measurable parameter, e.g.,immune attack, can be evaluated in an animal model system predictive ofefficacy in transplant rejection or autoimmune disorders. Alternatively,this property of a composition can be evaluated by examining the abilityof the compound to inhibit, such inhibition in vitro by assays known tothe skilled practitioner.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Also within the scope of the invention is a kit comprising a therapeuticcompound described herein. The kit can include one or more otherelements including: instructions for use; other reagents, e.g., a label,a therapeutic agent, or an agent useful for chelating, or otherwisecoupling, a therapeutic molecule to a label or other therapeutic agent,or a radioprotective composition; devices or other materials forpreparing the therapeutic molecule for administration; pharmaceuticallyacceptable carriers; and devices or other materials for administrationto a subject.

Combinations

The proteins described herein can also be administered in conjunctionwith other agents useful for treating the condition with which thepatient is suffering from. Examples of such agents include bothproteinaceous and non-proteinaceous drugs. When multiple therapeuticsare co-administered, dosages may be adjusted accordingly, as isrecognized in the pertinent art. “Co-administration” and combinationtherapy are not limited to simultaneous administration, but also includetreatment regimens in which a T-reg-selective IL-2 protein isadministered at least once during a course of treatment that involvesadministering at least one other therapeutic agent to the patient.

In some embodiments, a T-reg-selective IL-2 protein is administered incombination with an inhibitor of the PI3-K/AKT/mTOR pathway, e.g.,rapamycin (rapamune, sirolimus). Inhibitors of this pathway incombination with IL-2 favor T-reg enrichment. In some embodiments, theIL-2 protein is administered without another therapeutic that is notdirectly fused or attached to the IL-2 protein.

Therapeutic Methods

“Treatment” of any disease mentioned herein encompasses an alleviationof at least one symptom of the disease, a reduction in the severity ofthe disease, or the delay or prevention of disease progression to moreserious symptoms that may, in some cases, accompany the disease or to atleast one other disease. Treatment need not mean that the disease istotally cured. A useful therapeutic agent needs only to reduce theseverity of a disease, reduce the severity of symptom(s) associated withthe disease or its treatment, or delay the onset of more serioussymptoms or a more serious disease that can occur with some frequencyfollowing the treated condition. For example, if the disease is aninflammatory bowel disease, a therapeutic agent may reduce the number ofdistinct sites of inflammation in the gut, the total extent of the gutaffected, reduce pain and/or swelling, reduce symptoms such as diarrhea,constipation, or vomiting, and/or prevent perforation of the gut. Apatient's condition can be assessed by standard techniques such as anx-ray performed following a barium enema or enteroclysis, endoscopy,colonoscopy, and/or a biopsy. Suitable procedures vary according to thepatient's condition and symptoms.

-   In some embodiments, the proteins are used to treat inflammatory    disorders. In some embodiments, the inflammatory disorder is    inflammation, autoimmune disease, atopic diseases, paraneoplastic    autoimmune diseases, cartilage inflammation, arthritis, rheumatoid    arthritis (e.g. active), juvenile arthritis, juvenile rheumatoid    arthritis, pauciarticular juvenile rheumatoid arthritis,    polyarticular juvenile rheumatoid arthritis, systemic onset juvenile    rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile    enteropathic arthritis, juvenile reactive arthritis, juvenile    Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy,    Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic    arthritis, juvenile scleroderma, juvenile systemic lupus    erythematosus, juvenile vasculitis, pauciarticular rheumatoid    arthritis, polyarticular rheumatoid arthritis, systemic onset    rheumatoid arthritis, ankylosing spondylitis, enteropathic    arthritis, reactive arthritis, Reiter's Syndrome, SEA Syndrome    (Seronegativity, Enthesopathy, Arthropathy Syndrome),    dermatomyositis, psoriatic arthritis, scleroderma, vasculitis,    myolitis, polymyolitis, dermatomyolitis, polyarteritis nodossa,    Wegener's granulomatosis, arteritis, ploymyalgia rheumatica,    sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing    cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis,    guttate psoriasis, inverse psoriasis, pustular psoriasis,    erythrodermic psoriasis, dermatitis, atopic dermatitis, dermatitis    herpetiformis, Behcet's disease, including but not limited to the    effects on the skin, alopecia, alopecia areata, alopecia totalis,    atherosclerosis, lupus, Still's disease, Systemic Lupus    Erythematosus (SLE) (e.g. active), myasthenia gravis, inflammatory    bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac    disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis,    rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilic    bronchitis, Guillain-Barre disease, Type I diabetes mellitus,    thyroiditis(e.g., Graves' disease), Addison's disease, Raynaud's    phenomenon, autoimmune hepatitis, graft versus host disease, steroid    refractory chronic graft versus host disease, transplantation    rejection(e.g. kidney, lung, heart, skin, and the like), kidney    damage, hepatitis C-induced vasculitis, spontaneous loss of    pregnancy, alopecia, vitiligo, focal segmental glomerulosclerosis    (FSGS), Minimal Change Disease, Membranous Nephropathy, ANCA    Associated Glomerulonephropathy, Membranoproliferative    Glomerulonephritis, IgA Nephropathy, lupus nephritis, and the like.    In some embodiments, the proteins are used to treat steroid    refractory chronic graft versus host disease. In some embodiments,    the proteins are used to treat active systemic lupus erythematosus.    In some embodiments, the proteins are used to treat active    rheumatoid arthritis.

In some embodiments, the methods comprise administering a pharmaceuticalcomposition comprising the proteins described herein to the subject. Insome embodiments, the subject is a subject in need thereof. Any of theabove-described therapeutic proteins can be administered in the form ofa compositions (e.g. pharmaceutical compositions) that are describedherein. For example, a composition may comprise an IL-2 protein asdescribed herein plus a buffer, an antioxidant such as ascorbic acid, alow molecular weight polypeptide (such as those having less than 10amino acids), a protein, amino acids, carbohydrates such as glucose,sucrose, or dextrins, chelating agent such as EDTA, glutathione, and/orother stabilizers, excipients, and/or preservatives. The composition maybe formulated as a liquid or a lyophilizate. Further examples ofcomponents that may be employed in pharmaceutical formulations arepresented in Remington's Pharmaceutical Sciences, 16.sup.th Ed., MackPublishing Company, Easton, Pa., (1980) and others as described herein.

To treat the disease of interest, the compositions comprisingtherapeutic molecules described herein can be administered by anyappropriate method including, but not limited to, parenteral, topical,oral, nasal, vaginal, rectal, or pulmonary (by inhalation)administration. If injected, the composition(s) can be administeredintra-articularly, intravenously, intraarterially, intramuscularly,intraperitoneally, or subcutaneously by bolus injection or continuousinfusion. Localized administration, that is, at the site of disease, iscontemplated, as are transdermal delivery and sustained release fromimplants, skin patches, or suppositories. Delivery by inhalationincludes, for example, nasal or oral inhalation, use of a nebulizer,inhalation in aerosol form, and the like. Administration via asuppository inserted into a body cavity can be accomplished, forexample, by inserting a solid form of the composition in a chosen bodycavity and allowing it to dissolve. Other alternatives include eyedrops,oral preparations such as pills, lozenges, syrups, and chewing gum, andtopical preparations such as lotions, gels, sprays, and ointments. Inmost cases, therapeutic molecules that are polypeptides can beadministered topically or by injection or inhalation.

In the performance of the methods of treatment, the therapeuticmolecules described above can be administered as described herein andabove. For example, the composition can be administered at any dosage,frequency, and duration that can be effective to treat the conditionbeing treated. The dosage depends on the molecular nature of thetherapeutic molecule and the nature of the disorder being treated.Treatment may be continued as long as necessary to achieve the desiredresults. Therapeutic molecules of the invention can be administered as asingle dosage or as a series of dosages given periodically, includingmultiple times per day, daily, every other day, twice a week, threetimes per week, weekly, every other week, and monthly dosages, amongother possible dosage regimens. The periodicity of treatment may or maynot be constant throughout the duration of the treatment. For example,treatment may initially occur at weekly intervals and later occur everyother week. Treatments having durations of days, weeks, months, or yearsare encompassed by the invention. Treatment may be discontinued and thenrestarted. Maintenance doses may or may not be administered after aninitial treatment.

Dosage may be measured as milligrams per kilogram of body weight (mg/kg)or as milligrams per square meter of skin surface (mg/m²) or as a fixeddose, irrespective of height or weight. All of these are standard dosageunits in the art. A person's skin surface area is calculated from herheight and weight using a standard formula.

Also provided herein are methods of promoting stimulating STAT5phosphorylation in T regulatory cells. In some embodiments, the methodscomprise administering to a subject in need thereof a therapeuticallyeffective amount of a peptide described herein or a pharmaceuticalcomposition comprising the same.

As used herein, the phrase “in need thereof” means that the subject(animal or mammal) has been identified as having a need for theparticular method or treatment. In some embodiments, the identificationcan be by any means of diagnosis. In any of the methods and treatmentsdescribed herein, the animal or mammal can be in need thereof. In someembodiments, the animal or mammal is in an environment or will betraveling to an environment in which a particular disease, disorder, orcondition is prevalent.

Unless defined otherwise, all technical and scientific terms have thesame meaning as is commonly understood by one of ordinary skill in theart to which the embodiments disclosed belongs.

As used herein, the terms “a” or “an” means that “at least one” or “oneor more” unless the context clearly indicates otherwise.

As used herein, the term “about” means that the numerical value isapproximate and small variations would not significantly affect thepractice of the disclosed embodiments. Where a numerical limitation isused, unless indicated otherwise by the context, “about” means thenumerical value can vary by ±10% and remain within the scope of thedisclosed embodiments.

As used herein, the term “individual” or “subject,” or “patient” usedinterchangeably, means any animal, including mammals, such as mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,or primates, such as humans.

As used herein, the terms “comprising” (and any form of comprising, suchas “comprise”, “comprises”, and “comprised”), “having” (and any form ofhaving, such as “have” and “has”), “including” (and any form ofincluding, such as “includes” and “include”), or “containing” (and anyform of containing, such as “contains” and “contain”), are inclusive oropen-ended and do not exclude additional, unrecited elements or methodsteps. Any step or composition that uses the transitional phrase of“comprise” or “comprising” can also be said to describe the same withthe transitional phase of “consisting of” or “consists.”

As used herein, the term “contacting” means bringing together of twoelements in an in vitro system or an in vivo system. For example,“contacting” a peptide or composition described herein with a T-reg cellor with an individual or patient or cell includes the administration ofthe compound to an individual or patient, such as a human, as well as,for example, introducing a compound into a sample containing a cellularor purified preparation containing the T-reg cell.

As used herein, the term “fused” or “linked” when used in reference to aprotein having different domains or heterologous sequences means thatthe protein domains are part of the same peptide chain that areconnected to one another with either peptide bonds or other covalentbonding. The domains or section can be linked or fused directly to oneanother or another domain or peptide sequence can be between the twodomains or sequences and such sequences would still be considered to befused or linked to one another. In some embodiments, the various domainsor proteins provided for herein are linked or fused diretctly to oneanother or a linker sequences, such as the glycine/serine sequencesdescribed herein link the two domains together.

In some embodiments, embodiments provided herein also include, but arenot limited to:

-   1. A peptide comprising an amino acid sequence of SEQ ID NO: 1,    wherein the peptide comprises a mutation at position 73, 76, 100, or    138.-   2. The peptide of embodiment 1, wherein the mutation is a L to I    mutation at position 73, 76, 100, or 138.-   3. The peptide of embodiment 1, wherein the peptide further    comprises a mutation at one or more of positions 49, 51, 55, 57, 68,    89, 91, 94, 108, and 145.-   4. The peptide of embodiment 1, further comprising a mutation at one    or more of positions E35, H36, Q42, D104, E115, or Q146 or 1, 2, 3,    4, 5, or each of E35, H36, Q42, D104, E115, or Q146 is wild-type.-   5. The peptide of embodiment 4, wherein the mutation is one or more    of E35Q, H36N, Q42E, D104N, E115Q, or Q146E.-   6. The peptide of any one of embodiments 1-5, wherein the peptide    comprises a N49S mutation.-   7. The peptide of any one of embodiments 1-6, wherein the peptide    comprises a Y51S or a Y51H mutation.-   8. The peptide of any one of embodiments 1-7, wherein the peptide    comprises a K55R mutation.-   9. The peptide of any one of embodiments 1-8, wherein the peptide    comprises a T57A mutation.-   10. The peptide of any one of embodiments 1-9, wherein the peptide    comprises a K68E mutation.-   11. The peptide of any one of embodiments 1-10, wherein the peptide    comprises a V89A mutation.-   12. The peptide of any one of embodiments 1-11, wherein the peptide    comprises a N91R mutation.-   13. The peptide of any one of embodiments 1-12, wherein the peptide    comprises a Q94P mutation.-   14. The peptide of any one of embodiments 1-13, wherein the peptide    comprises a N108D or a N108R mutation.-   15. The peptide of any one of embodiments 1-14, wherein the peptide    comprises a C145A or C145S mutation.-   15.1. The peptide of any one of the embodiments 1-15, wherein the    peptide comprises V89A, Q94P, N108R or N108D, and one or more of    L73I, L76I, L100I, L118I mutations, and optionally C145A or C145S    mutation.-   16. A peptide comprising an amino acid sequence of SEQ ID NO: 2,    wherein the peptide comprises a mutation at position 53, 56, 80, or    118.-   17. The peptide of embodiment 16, wherein the mutation is a L to I    mutation at position 53, 56, 80, or 118.-   18. The peptide of embodiments 16 or 17, wherein the peptide further    comprises a mutation at one or more of positions 29, 31, 35, 37, 48,    69, 71, 74, 88, and 125.-   19. The peptide of embodiment 16, further comprising a mutation at    one or more of positions E15, H16, Q22, D84, E95, or Q126 or 1, 2,    3, 4, 5, or each of positions E15, H16, Q22, D84, E95, or Q126 is    wild-type.-   20. The peptide of embodiment 19, wherein the mutation is one or    more of E15Q, H16N, Q22E, D84N, E95Q, or Q126E.-   21. The peptide of any one of embodiments 16-20, wherein the peptide    comprises a N29S mutation.-   22. The peptide of any one of embodiments 16-21, wherein the peptide    comprises a Y31S or a Y51H mutation.-   23. The peptide of any one of embodiments 16-22, wherein the peptide    comprises a K35R mutation.-   24. The peptide of any one of embodiments 16-23, wherein the peptide    comprises a T37A mutation.-   25. The peptide of any one of embodiments 16-24, wherein the peptide    comprises a K48E mutation.-   26. The peptide of any one of embodiments 16-25, wherein the peptide    comprises a V69A mutation.-   27. The peptide of any one of embodiments 16-26, wherein the peptide    comprises a N71R mutation.-   28. The peptide of any one of embodiments 16-27, wherein the peptide    comprises a Q74P mutation.-   29. The peptide of any one of embodiments 16-28, wherein the peptide    comprises a N88D or a N88R mutation.-   30. The peptide of any one of embodiments 16-29, wherein the peptide    comprises a C125A or C125S mutation.-   31. The peptide of any one of the embodiments 16-30, wherein the    peptide comprises V69A, Q74P, N88R or N88D, and one or more of L53I,    L56I, L80I, L118I mutations, and optionally C125A or C125S mutation.-   32. The peptide of any of the preceding embodiments, wherein the    peptide does not comprise a mutation that corresponds to positions    30, 31, and/or 35.-   33. The peptide of any of the preceding embodiments, further    comprising a Fc peptide.-   33A. The peptide of embodiment 33, wherein the Fc peptide comprises    a mutation at position, using the Kabat numbering L247, L248, and    G250, or using the EU numbering at positions L234, L235, and G237.-   33B. The peptide of embodiment 33, wherein the Fc peptide comprises    the following mutations: L247A, L248A, and G250A (Kabat Numbering)    or L234A L235A, and G237A (EU Numbering).-   34. The peptide of embodiment 33, wherein the Fc peptide comprises    the sequence of SEQ ID NO: 8 or SEQ ID NO: 15.-   35. The peptide of any of the preceding embodiments, further    comprising a linker peptide that links the peptide of SEQ ID NO: 1    or SEQ ID NO: 2 and the Fc peptide.-   36. The peptide of embodiment 35, wherein the linker comprises a    sequence of GGGGSGGGGSGGGGSGGGGS or GGGGSGGGGSGGGGS.-   37. A peptide of any of the preceding embodiments, wherein the    peptide comprises a sequence of SEQ ID NO: 17-42.-   38. A peptide comprising an amino acid sequence of SEQ. ID. NO: 27.-   39. The peptide of embodiment 38, further comprising a N-terminal    leader peptide having the sequence of SEQ. ID. No: 7.-   40. The peptide of embodiment 1, further comprising a linker peptide    at the C-terminus.-   41. The peptide of embodiment 40, wherein the linker peptide    comprises the sequence of (GGGGS)_(n), wherein n is 1, 2, 3, or 4.-   42. The peptide of embodiment 41, wherein n is 1.-   43. The peptide of embodiment 41, wherein n is 2.-   44. The peptide of embodiment 41, wherein n is 3.-   45. The peptide of embodiment 41, wherein n is 4.-   46. The peptide of any one of embodiments 48-45, further comprising    a Fc peptide.-   47. The peptide of embodiment 46, wherein the Fc peptide comprises    the sequence of SEQ ID NO: 8 or SEQ ID NO: 15.-   48. The peptide of embodiment 47, further comprising a leader    peptide having the sequence of SEQ. ID. No: 7 at the N-terminus.-   49. The peptide of embodiment 46, wherein the Fc peptide is at the    C-terminus of the peptide comprising SEQ ID NO: 27.-   50. The peptide of embodiment 46, wherein the Fc peptide is at the    N-terminus of the peptide comprising SEQ ID NO: 27.-   51. The peptide of embodiment 1, further comprising a linker peptide    that links the peptide of SEQ ID NO: 27 and a Fc peptide.-   52. The peptide of embodiment 51, wherein the linker peptide is    (GGGGS)_(n), wherein n is 1, 2, 3, or 4.-   53. The peptide of embodiment 52, wherein n is 4.-   54. The peptide of embodiments 51-53, wherein the Fc peptide    comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 15.-   55. The peptide of embodiments 51-54, wherein the Fc peptide is at    the C-terminus of the peptide comprising SEQ ID NO: 27.-   56. The peptide of embodiments 51-54, wherein the Fc peptide is at    the N-terminus of the peptide comprising SEQ ID NO: 27.-   57. The peptide of embodiment 14, wherein the peptide comprises a    peptide comprising the sequence of SEQ ID NOs: 37, 38, 39, or 40.-   58. A pharmaceutical composition comprising a peptide of any one of    the preceding embodiments.-   59. A method of activating T regulatory cells, the method comprising    contacting a T regulatory cell with a peptide of any one of    embodiments 1-57 or the pharmaceutical composition of embodiment 58.-   60. A method of treating an inflammatory disorder in a subject, said    method comprising administering to a subject in need thereof a    therapeutically effective amount of a peptide of any one of    embodiments 1-57 or the pharmaceutical composition of embodiment 58.-   61. The method of embodiment 60, wherein the inflammatory disorder    is inflammation, autoimmune disease, atopic diseases, paraneoplastic    autoimmune diseases, cartilage inflammation, arthritis, rheumatoid    arthritis, juvenile arthritis, juvenile rheumatoid arthritis,    pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile    rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis,    juvenile ankylosing spondylitis, juvenile enteropathic arthritis,    juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA    Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),    juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile    scleroderma, juvenile systemic lupus erythematosus, juvenile    vasculitis, pauciarticular rheumatoid arthritis, polyarticular    rheumatoid arthritis, systemic onset rheumatoid arthritis,    ankylosing spondylitis, enteropathic arthritis, reactive arthritis,    Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy,    Arthropathy Syndrome), dermatomyositis, psoriatic arthritis,    scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis,    polyarteritis nodossa, Wegener's granulomatosis, arteritis,    ploymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary    sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis,    plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular    psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis,    atherosclerosis, lupus, Still's disease, Systemic Lupus    Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease    (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple    sclerosis (MS), asthma, COPD, rhinosinusitis, rhinosinusitis with    polyps, eosinophilic esophogitis, eosinophilic bronchitis,    Guillain-Barre disease, Type I diabetes mellitus, thyroiditis(e.g.,    Graves' disease), Addison's disease, Raynaud's phenomenon,    autoimmune hepatitis, graft versus host disease (GVHD), steroid    refractory chronic graft versus host disease, transplantation    rejection (e.g. kidney, lung, heart, skin, and the like), kidney    damage, hepatitis C-induced vasculitis, spontaneous loss of    pregnancy, alopecia, vitiligo, focal segmental glomerulosclerosis    (FSGS), Minimal Change Disease, Membranous Nephropathy, ANCA    Associated Glomerulonephropathy, Membranoproliferative    Glomerulonephritis, IgA Nephropathy, lupus nephritis, and the like.-   62. A method of promoting or stimulating STAT5 phosphorylation in T    regulatory cells, said method administering to a subject in need    thereof a therapeutically effective amount of a peptide of any one    of embodiments 1-57 or the pharmaceutical composition of embodiment    58.-   63. Use of a peptide of any one of embodiments 1-57 or the    pharmaceutical composition of embodiment 58 in the preparation of a    medicament for the treatment of an inflammatory disorder.-   64. The use of embodiment 63, wherein the inflammatory disorder is    inflammation, autoimmune disease, atopic diseases, paraneoplastic    autoimmune diseases, cartilage inflammation, arthritis, rheumatoid    arthritis, juvenile arthritis, juvenile rheumatoid arthritis,    pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile    rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis,    juvenile ankylosing spondylitis, juvenile enteropathic arthritis,    juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA    Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),    juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile    scleroderma, juvenile systemic lupus erythematosus, juvenile    vasculitis, pauciarticular rheumatoid arthritis, polyarticular    rheumatoid arthritis, systemic onset rheumatoid arthritis,    ankylosing spondylitis, enteropathic arthritis, reactive arthritis,    Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy,    Arthropathy Syndrome), dermatomyositis, psoriatic arthritis,    scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis,    polyarteritis nodossa, Wegener's granulomatosis, arteritis,    ploymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary    sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis,    plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular    psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis,    atherosclerosis, lupus, Still's disease, Systemic Lupus    Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease    (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple    sclerosis (MS), asthma, COPD, rhinosinusitis, rhinosinusitis with    polyps, eosinophilic esophogitis, eosinophilic bronchitis,    Guillain-Barre disease, Type I diabetes mellitus, thyroiditis(e.g.,    Graves' disease), Addison's disease, Raynaud's phenomenon,    autoimmune hepatitis, graft versus host disease (GVHD), steroid    refractory chronic graft versus host disease, transplantation    rejection(e.g. kidney, lung, heart, skin, and the like), kidney    damage, hepatitis C-induced vasculitis, spontaneous loss of    pregnancy, alopecia, vitiligo, focal segmental glomerulosclerosis    (FSGS), Minimal Change Disease, Membranous Nephropathy, ANCA    Associated Glomerulonephropathy, Membranoproliferative    Glomerulonephritis, IgA Nephropathy, lupus nephritis, and the like.-   65. A nucleic acid molecule encoding a peptide of any one of    embodiments 1-57.-   66. A vector comprising the nucleic acid molecule of embodiment 65.-   67. A plasmid comprising the nucleic acid molecule of embodiment 65.-   68. A cell comprising the nucleic acid molecule of embodiment 65.-   69. A cell comprising the plasmid of embodiment 67.-   70. A cell comprising the vector of embodiment 66.-   71. A cell comprising or expressing a peptide of any one of    embodiments 1-57 or a peptide as described herein.

The following examples are illustrative, but not limiting, of thecompounds, compositions and methods described herein. Other suitablemodifications and adaptations known to those skilled in the art arewithin the scope of the following embodiments.

EXAMPLES Example 1

A therapeutic composition comprising a protein of SEQ ID NOs: 11, 12,13, or 14 is administered to a subject suffering from IBD. The subject'simmune system is down-regulated and the symptoms of the IBD arealleviated.

Example 2

A therapeutic composition comprising a protein of SEQ ID NOs: 3, 4, 5,or 6, with or without the leader sequence, is administered to a subjectsuffering from IBD. The subject's immune system is down-regulated andthe symptoms of the IBD are alleviated.

Example 3 Generation of IL-Muteins

A pTT5 vector containing the single gene encoding the human IL-2Mpolypeptide fused N-terminally (SEQ ID NO: 40) or C-terminally (SEQ IDNO: 41) to human IgG1 Fc domain was transfected into HEK293 Expi cells.After 5-7 days, cell culture supernatants expressing IL-2Ms wereharvested, and clarified by centrifugation and filtered through a 0.22um filtration device. IL-2Ms were captured on proA resin. The resin waswashed with PBS pH 7.4 and the captured protein was eluted using 0.25%acetic acid pH 3.5, with neutralization using a tenth volume of 1M TrispH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH7, and analyzed by size exclusion chromatography on a Superdex 2003.2/300 column. Analysis of 5 ug of purified material by reducing andnon-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted. The IL-2Mswere expressed at over 10 mg/L, and were over 95% monodispersed afterpurification as shown by size exclusion chromatography andreducing/non-reducing SDS-PAGE.

Example 4 IL-2M Molecules can Bind CD25

An immunosorbent plate was coated with CD25 at a concentration of 0.5μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wellswere washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer)three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4(block buffer) for two hours at room temperature. After three washeswith wash buffer IL-2M molecules were diluted to eleven -two fold serialdilution in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) with2 nM being the highest concentration. The diluted material was added tothe CD25 coated plate at 75 ul/well for 1 hour at room temperature.After three washes with wash buffer, a goat biotinylated anti-IL-2polyclonal antibody, diluted to 0.05 μg/mL in assay buffer, was added tothe plate at 75 ul/well for 1 hr at room temperature. After three washeswith wash buffer streptavidin HRP diluted in assay buffer at 1:5000 wasadded to the plate at 75 ul/well for 15 minutes at room temperature.After three washes with wash buffer and 1 wash with wash buffer (with notween-20), the assay was developed with TMB, and stopped with 1N HCL. OD450 nm was measured. The experiment included appropriate controls fornon-specific binding of IL-2M molecules to the plate/block in theabsence of CD25 and a negative control molecule that is unable to bindCD25.

The results indicate that at concentrations of 2 nM-1.9 pM, IL-2Mmolecules are able to bind CD25 with sub nanomolar EC50s. Additionally,there was no detection of any compound at any concentration tested, whenCD25 was not present on the plate surface, indicating none of the testcompounds were interacting non-specifically with the plate surface (datanot shown).

Example 5

In Vitro P-STAT5 Assay To Determine Potency and Selectivity of IL-2MMolecules. Peripheral blood mononuclear cells (PBMCs) were preparedusing FICOLL-PAQUE Premium and Sepmate tubes from freshly isolatedheparinized human whole blood. PBMCs were cultured in 10% fetal bovineserum RPMI medium in the presence of wild-type IL-2 or IL-2M of Example12 for 20 minutes and then fixed for 10 minutes with BD Cytofix.

Fixed cells were sequentially permeabilized with BD Perm III and thenBioLegend FOXP3 permeabilization buffer. After blocking with human serumfor 10 minutes, cells were stained for 30 minutes with antibodies forphospho-STAT5 FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 and thenacquired on an Attune NXT with plate reader. The IL-2M of SEQ ID NO: 23potently and selectively induces STAT5 phosphorylation in Tregs but notTeffs.

Example 6 Immunogenicity of IL-2 Muteins

IL-2 Mutein Mutant sequences were analyzed using the NetMHCIIPan 3.2software, which can be found at www “dot” cbs “dot” dtu “dot”dk/services/NetMHCIIpan/. Artificial neural networks were used todetermine peptide affinity to MHC class II alleles. In that analysis,9-residue peptides with potentially direct interaction with the MHCclass II molecules were recognized as binding cores. Residues adjacentto binding cores, with potential to influence the binding indirectly,were also examined as masking residues. Peptides comprising both thebinding cores and masking residues were marked as strong binders whentheir predicted K_(D) to the MHC class II molecule was lower than 50 nM.Strong binders have a greater chance of introducing T cellimmunogenicity.

A total of 9 MHCII alleles that are highly represented in North Americaand Europe were included in the in silico analysis. The panel of IL-2M(IL-2 muteins) molecules tested included the IL-2 Muteins with L53I,L56I, L80I, or L118I mutations. Only MHCII alleles DRB1_1101, DRB1_1501,DRB1_0701, and DRB1_0101 yielded hits with any of the moleculesassessed. The peptide hits for DRB_1501 were identical between allconstructs tested including wild-type IL-2 with the C125S mutation. Theaddition of L80I removes 1 T cell epitope for DRB1-0101[ALNLAPSKNFHLRPR] and modestly reduces the affinity of two other T cellepitopes [EEALNLAPSKNFHLR and EALNLAPSKNFHLRP]. For WWII alleleDRB1-0701, L80I removes 1 T cell epitope [EEALNLAPSKNFHLR]. Therefore,the data demonstrates that a IL-2 mutein comprising the L80I mutationshould be less immunogenic, which is a surprising and unexpected resultfrom the in silico analysis.

Example 7 Generation of Additional IL-2 Muteins

A pTT5 vector containing the single gene encoding the single IL-2M (IL-2mutein) SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (andIL-2M control; SEQ ID NO: 34) polypeptide with human IL-2M or IL-2Mfused N-terminally of human IgG1 Fc domain was transfected into HEK293Expi cells. After 5-7 days, cell culture supernatants expressing SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL-2M control;SEQ ID NO: 34) were harvested, and clarified by centrifugation andfiltration through a 0.22 um filtration device. SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34)were captured on proA resin. The resin was washed with PBS pH 7.4 andthe captured protein was eluted using 0.25% acetic acid pH 3.5, withneutralization using a tenth volume of 1M Tris pH 8.0. The protein wasbuffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by sizeexclusion chromatography on a Superdex 200 3.2/300 column. Analysis of 5ug of purified material by reducing and non-reducing SDS-PAGE on aBis-Tris 4-12% gel was conducted.

IL-2Ms SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (andIL-2M control; SEQ ID NO: 34) expressed at over 45 mg/L, and were over95% monodispersed after purification as shown by size exclusionchromatography and reducing/non-reducing SDS-PAGE.

Example 8 IL-2Ms can Bind CD25

An immunosorbent plate was coated with CD25 at a concentration of 0.5μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wellswere washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer)three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4(block buffer) for two hours at room temperature. After three washeswith wash buffer IL-2Ms SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQID NO: 40 were diluted to eleven -two fold serial dilution in PBScontaining 1% BSA and 0.05% Tween-20 (assay buffer) with 2 nM being thehighest concentration. The diluted material was added to the CD25 coatedplate at 75 ul/well for 1 hour at room temperature. After three washeswith wash buffer, a goat biotinylated anti-IL-2 polyclonal antibody,diluted to 0.05 μg/mL in assay buffer, was added to the plate at 75ul/well for 1 hr at room temperature. After three washes with washbuffer high sensitivity streptavidin HRP diluted in assay buffer at1:5000 was added to the plate at 75 ul/well for 15 minutes at roomtemperature. After three washes with wash buffer and 1 wash with washbuffer (with no tween-20), the assay was developed with TMB, and stoppedwith 1N HCL. OD 450 nm was measured. The experiment included appropriatecontrols for non-specific binding of the molecules to the plate/block inthe absence of CD25. The results indicate that at concentrations of 2nM-1.9 pM, the muteins of Example 7 were able to bind CD25 with subnanomolar EC50s. Additionally, there was no detection of any compound atany concentration tested, when CD25 was not present on the platesurface, indicating none of the test compounds were interactingnon-specifically with the plate surface. Thus, the muteins of Example 7can bind to CD25.

Example 9 IL-2 Muteins of Example 7 are Potent and Selective

Peripheral blood mononuclear cells (PBMCs) were prepared usingFICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinizedhuman whole blood. PBMCs were cultured in 10% fetal bovine serum RPMImedium in the presence of wild-type IL-2 or the muteins of Example 7 for20 minutes and then fixed for 10 minutes with BD Cytofix. Fixed cellswere sequentially permeabilized with BD Perm III and then BioLegendFOXP3 permeabilization buffer. After blocking with human serum for 10minutes, cells were stained for 30 minutes with antibodies forphospho-STAT5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (allBD) and then acquired on an Attune NXT with plate reader. The IL-2muteins of Example 7 were found to be potent and have selectivityagainst Treg versus Teff. The mutein comprising the L118I mutation wasfound to have increased activity and selectivity as compared to theother muteins.

Example 10 IL-2 Muteins Expand Tregs in Humanized Mice

NSG mice humanized with human CD34+ hematopoietic stem cells werepurchased from Jackson Labs. On days 0 and 7, the mice were dosedsubcutaneously with lug IL-2Mutein SEQ ID NO: 34 or other IL-2 muteinsSEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. On Day 7,mice were euthanized and whole blood and spleens were collected. Wholeblood was aliquoted into a 96 well deep well plate and fixed for 10minutes using BD Fix Lyse. Splenocytes were isolated using 70 um filters(BD) and red blood cells were lysed using RBC lysis buffer fromBioLegend. After washing with 2% fetal bovine serum PBS, splenocyteswere labeled with near infrared live dead stain (Invitrogen) for 20minutes and then fixed for 20 minutes using BioLegend fixation buffer.Both whole blood cells and splenocytes were then permeabilized usingBioLegend FOXP3 permeabilization buffer, blocked with human serum andstained for 30 minutes with antibodies against human CD8a FITC (BL),human CD25 PE (BD), human FOXP3 AF647 (BD) CD4 PerCP Cy5.5 (BD), humanSiglec-8 PE Cy7 (BL), human CD3 BV421 (BL), human CD45 BV605 (BL), humanCD56 BV785 (BL) and mouse CD45 (BV711) and acquired on an Attune NXTwith plate loader.

Compared to vehicle control, IL-2Ms SEQ ID NO: 37 and SEQ ID NO: 38 andSEQ ID NO: 39 and SEQ ID NO: 40 selectively induced human Tregs in mousespleens and whole blood in humanized mice. There were no significantchanges in the frequencies of human CD56pos NK cells, CD3pos T cells,CD8pos cytotoxic T lymphocytes, CD4pos helper T cells or CD25lo/FOXP3negT effectors. These results demonstrate that the IL-2 muteins increasethe frequency of regulatory T cells.

Example 11 Durability of Signaling Induced by IL-2 Muteins

Peripheral blood mononuclear cells (PBMCs) were prepared usingFICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinizedhuman whole blood. PBMCs were cultured in 10% fetal bovine serum RPMImedium in the presence of IL-2M for 60 minutes. Cells were then wash 3times and incubated for an additional 3 hours. Cells were then fixed for10 minutes with BD Cytofix. Fixed cells were sequentially permeabilizedwith BD Perm III and then BioLegend FOXP3 permeabilization buffer. Afterblocking with human serum for 10 minutes, cells were stained for 30minutes with antibodies for phospho-STAT5 FITC, CD25 PE, FOXP3 AF647 andCD4 PerCP Cy5.5 and then acquired on an Attune NXT with plate reader.All four IL-2 muteins of Example 19 induced durable signaling in Tregbut not in Teff as compared to the control. SEQ ID NO: 40 is superior toSEQ ID NO: 39, SEQ ID NO: 38 or SEQ ID NO: 37. These results demonstratethat the IL-2 can induce durable and selective signaling in Treg whichshould lead to greater Treg expansion in vivo and permit less frequentdosing to achieve Treg expansion.

The examples provided for herein demonstrate the surprising andunexpected result that a IL-2 mutein can function to selectively andpotently activate Tregs over Teffs, which demonstrates that themolecules can be used to treat or ameliorate the conditions describedherein. The IL-2 muteins, as provided for herein, can also be generatedand used with or without being fused to a Fc domain or a linker asprovided for herein.

The embodiments has been described with reference to specific examples.These examples are not meant to limit the embodiments in any way. It isunderstood for purposes of this disclosure, that various changes andmodifications may be made that are well within the scope of the presentdisclosure. Numerous other changes may be made which will readilysuggest themselves to those skilled in the art and which are encompassedin the spirit of the invention disclosed herein and as defined in theappended claims.

Example 12 Muteins Exhibit Overall POI and Lower Aggregation

IL-2 muteins with the mutations of V69A, Q74P, N88D, and C125S and oneof the following mutations L53I, L56I, L80I, or L118I linked to a Fcregion comprising L234A, L235A, and G237A mutations as provided forherein were expressed in a pTT5 vector by transfecting the vector intoHEK293 Expi cells. The IL-2 mutein was linked to the N-terminus of theFc region with 4 GGGGS repeats. After 5-7 days, cell culturesupernatants expressing the different IL-2 muteins were harvested, andclarified by centrifugation and filtration through a 0.22 um filtrationdevice. The IL-2 muteins were captured on proA resin. The resin waswashed with PBS pH 7.4 and the captured protein was eluted using 0.25%acetic acid pH 3.5, with neutralization using a tenth volume of 1M TrispH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH7, and analyzed by size exclusion chromatography on an AdvanceBio SECcolumn for percent peak of interest (POI). The results demonstrated thatthe different muteins were expressed at over 60 mg/L. However, it wassurprisingly found that muteins with the L80I or L118I mutation weregreater than 90% monodispersed while muteins with the L53I or L56Imutations were not as shown by size exclusion chromatography. Thus, themuteins with the L80I or L118I substitution had less aggregation. Thedifferences in aggregation amongst the four molecules (IL-2 muteinscomprising L80I, L118I, L53I, and L56I) were surprising due to the typeof mutation that was being made. Therefore, the muteins with the L80I orthe L118I mutation have a surprising advantage over other muteins inthat it does not aggregate as much as other muteins.

Example 13 IL-2 Muteins Have Unexpected Increase in Potency

The muteins described in Example 12 were analyzed for potency in an invitro assay. Briefly, PBMCs were isolated from heparinized human wholeblood and stimulated with the different muteins at a concentration for30 min at 37 C. The stimulation was stopped by fixation. Afterpermeabilization, PBMCs were stained for intracellular FoxP3 andphospho-STAT5 levels and surface CD4 and CD25 expression and analyzed byflow cytometry. Regulatory T cells (Tregs) and effector T cells (Teffs)were gated as CD4+CD25hiFoxP3+ or CD4+CD251oFoxP3−, respectively. Thepercent of cells that stained positive for phospho-STAT5 is shown. Thisassay measures the ability of the muteins to specifically stimulateTregs without stimulating Teffs. A best-fit dose-response curve for eachtest article was used to calculate an EC50 value.

Surprisingly, the muteins with the mutations of L118I, L80I, L56I, orL53I had increased potency (stimulating Tregs) as compared to an IL-2mutein without any of these mutations. The IL-2 mutein without amutation of L118I, L80I, L56I, or L53I, but having the V69A, Q74P, andN88D mutations was approximately 51 pM. Each of the EC₅₀s for themuteins comprising one of L118I, L80I, L56I, or L53I had EC₅₀s ofapproximately 30, 40, 41, and 45, respectively. The differences in EC50for stimulating Tregs (with no changes in Teff stimulation) wassurprising and would not have been predicted for the muteins having oneof the mutations described in this example. The data can also beevaluated by comparing the ratio of the parent IL-2 muteins (comprisingV69A, Q74P, N88D, and C125S) to the muteins that also comprise one ofL118I, L80I, L56I, or L53I mutations. Using this ratio normalizes fordifferent cell populations that are used for different experiments.Using this ratio the L118I had an average increase of approximately 25%more potency (standard error of mean 0.16) as compared to the parentcontrol, whereas the other mutations had a decrease in activity ascompared to the parent control using this ratio.

The in vitro data was confirmed in vivo for the muteins having one ofL118I, L80I, L56I, or L53I mutations. L118I was found to be more potentthan a mutein without the L118I mutation in vivo. Briefly,Nod-Scid-IL-2Rgamma-deficient (NSG) mice reconstituted with human CD34+hematopoietic stem cells were injected subcutaneously with 1 microgramof the indicated test article or vehicle on days 0 and 7. On Day 11,mice were killed and blood was collected by cardiac puncture into tubescontaining heparin. Peripheral blood leukocytes (PBLs) were isolated bylysis of red blood cells and stained with antibodies reactive to thehuman markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56. The percent ofhuman regulatory T cells (Tregs, CD45+CD3+CD4+CD25+FoxP3+), activatedeffector T cells (act Teff, CD45+CD3+CD4+CD25+FoxP3−) and NK cells(CD45+CD56+) was determined by flow cytometry. The frequency of totalCD45+, total CD4+ and total CD8+ cells did not change. Similar resultswere observed in the spleens of mice. The in vivo potency as measured inthis assay of the IL-2 mutein with the L80I mutation was slightlyincreased as compared to a mutein without the L80I mutation and the invivo potency as measured in this assay of the muteins with either theL56I or the L53I mutation was about the same as a mutein without themutations. The muteins were N-terminal linked to a Fc region asdescribed herein with a 20 amino acid (GGGGS)₄ linker. That is thelinker connected the C-terminus of the IL-2 mutein to the N-terminus ofthe Fc region.

Example 14

N-terminal Fc Orientation with a 20 Amino Acid Linker is Most Effectiveat Stimulating Tregs. An IL-2 mutein with V69A, Q74P, and N88D was fusedto a Fc region comprising the mutations of L234A, L235A and G237Amutation using different lengths of GGGGS repeats. IL2-Mutein moleculesfused via the c-terminus of the mutein to the n-terminus of human IgG1Fc with linkers comprising 3 and 4 GGGGS repeats were tested todetermine if the length of the linker affected the potency of the IL-2mutein. A mutein fused via its n-terminus to the c-terminus of humanIgG1 Fc with a single GGGGS repeat was also tested. Briefly,Nod-Scid-IL-2Rgamma-deficient (NSG) mice reconstituted with human CD34+hematopoietic stem cells were injected subcutaneously with 1 microgramof the different Il-2 muteins with different linker lengths or vehicleon day 0. On Day 7, mice were sacrificed and blood was collected bycardiac puncture into tubes containing heparin. Peripheral bloodleukocytes (PBLs) were isolated by lysis of red blood cells and stainedwith antibodies reactive to the human markers CD45, CD3, CD8, CD4,FoxP3, CD25 and CD56. The percent of human regulatory T cells (Tregs,CD45+CD3+CD4+CD25+FoxP3+), activated effector T cells (act Teff,CD45+CD3+CD4+CD25+FoxP3−) and NK cells (CD45+CD56+) was determined byflow cytometry. The frequency of total CD45+, total CD4+ and total CD8+cells did not change. Similar results were observed in the spleens ofmice.

It was found that the mutein fused at the N-terminus of the human IgG1Fc with a linker comprising 4 GGGGS repeats was the most potent ascompared to a mutein with a linker that only had 3 GGGGS repeats or amutein fused at the c-terminus of the human IgG1 Fc with a single GGGGSrepeat. Additionally, although the protein with the 4 GGGGS repeats wasmore effective at expanding Tregs, the configuration did not trigger anydifferential expansion of CD56+ NK cells. It could not have beenpredicted that protein with N-terminally Fc fused mutein with the longerlinker would be the most potent and also not trigger any differentialexpansion of CD56+ NK cells.

Example 15 Treating Patients with Active Rheumatoid Arthritis

A pharmaceutical composition comprising a IL-2 mutein protein comprisinga sequence of SEQ ID NO: 37, 38, 39, or 40 are administered to patientswith active rheumatoid arthritis. The IL-2 muteins are found to beeffective in treating active rheumatoid arthritis in the patients.

Example 16 Treating Patients with Subjects with Active Systemic LupusErythematosus

A pharmaceutical composition comprising a IL-2 mutein protein comprisinga sequence of SEQ ID NO: 37, 38, 39, or 40 are administered to patientswith active systemic lupus erythematosus. The IL-2 muteins are found tobe effective in treating active systemic lupus erythematosus.

Example 17 Treating Patients with Subjects with Steroid RefractoryChronic Graft Versus Host Disease

A pharmaceutical composition comprising a IL-2 mutein protein comprisinga sequence of SEQ ID NO: 37, 38, 39, or 40 are administered to patientswith Steroid refractory chronic graft versus host disease. The IL-2muteins are found to be effective in treating steroid refractory chronicgraft versus host disease.

This specification contains numerous citations to patents, patentapplications, and publications. Each is hereby incorporated by referencefor all purposes.

What is claimed is:
 1. A protein consisting of an amino acid sequence ofSEQ ID NO:
 39. 2. A pharmaceutical composition comprising a protein ofclaim 1 and a pharmaceutically acceptable carrier.